Key Laboratory of Genetic Evolution & Animal Models, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China.
University of Chinese Academy of Sciences, Beijing, China.
Cell Mol Life Sci. 2024 Aug 22;81(1):366. doi: 10.1007/s00018-024-05413-x.
Chromosome stability is crucial for homeostasis of pluripotent stem cells (PSCs) and early-stage embryonic development. Chromosomal defects may raise carcinogenic risks in regenerative medicine when using PSCs as original materials. However, the detailed mechanism regarding PSCs chromosome stability maintenance is not fully understood.
Mouse embryonic stem cells (line D3) and human embryonic stem cells (line H9) were cultured under standard conditions. To confirm the loading of RetSat protein on mitotic chromosomes of PSCs, immunostaining was performed in PSCs spontaneous differentiation assay and iPSC reprogramming assay from mouse embryonic fibroblasts (MEFs), respectively. In addition, qPCR, immunoprecipitation, LC-MS/MS and immunoblotting were used to study the expression of RetSat, and interactions of RetSat with cohesin/condensin components. RNA sequencing and teratoma formation assay was conducted to evaluate the carcinogenic risk of mouse embryonic stem cells with RetSat deletion.
We reported a PSC high-expressing gene, RetSat, plays key roles in chromosome stabilization. We identified RetSat protein localizing onto mitotic chromosomes specifically in stemness positive cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found dramatic chromosome instability, e.g. chromosome bridging, lagging and interphase micronuclei in mouse and human ESCs when down regulating RetSat. RetSat knock-out mouse ESCs upregulated cancer associated gene pathways, and displayed higher tumorigenic capacities in teratoma formation assay. Mechanistically, we confirmed that RetSat interacts with cohesin/condensin components Smc1a and Nudcd2. RetSat deletion impaired the chromosome loading dosage of Smc1a, Smc3 and Nudcd2.
In summary, we reported RetSat to be a key stabilizer of chromosome condensation in pluripotent stem cells. This highlights the crucial roles of RetSat in early-stage embryonic development, and potential value of RetSat as an effective biomarker for assessing the quality of pluripotent stem cells.
染色体稳定性对于多能干细胞（PSCs）和早期胚胎发育的内稳态至关重要。当使用 PSCs 作为原始材料进行再生医学时，染色体缺陷可能会增加致癌风险。然而，PSCs 染色体稳定性维持的详细机制尚不完全清楚。
在标准条件下培养小鼠胚胎干细胞（D3 系）和人胚胎干细胞（H9 系）。为了确认 RetSat 蛋白在 PSCs 有丝分裂染色体上的加载情况，分别在 PSCs 自发分化实验和从小鼠胚胎成纤维细胞（MEFs）重编程的 iPSC 实验中进行免疫染色。此外，还使用 qPCR、免疫沉淀、LC-MS/MS 和免疫印迹来研究 RetSat 的表达及其与着丝粒/凝聚蛋白成分的相互作用。进行 RNA 测序和畸胎瘤形成实验来评估具有 RetSat 缺失的小鼠胚胎干细胞的致癌风险。
我们报道了一种 PSC 高表达基因 RetSat，它在染色体稳定中发挥关键作用。我们鉴定了 RetSat 蛋白特异性定位于有丝分裂染色体上，特别是在胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）等干性阳性细胞中。我们发现，当下调 RetSat 时，小鼠和人 ESC 中出现严重的染色体不稳定，例如染色体桥接、滞后和有丝分裂中期微核。RetSat 敲除的小鼠 ESC 上调了癌症相关基因途径，并在畸胎瘤形成实验中显示出更高的致瘤能力。从机制上讲，我们证实 RetSat 与着丝粒/凝聚蛋白成分 Smc1a 和 Nudcd2 相互作用。RetSat 缺失会损害 Smc1a、Smc3 和 Nudcd2 的染色体加载剂量。
总之，我们报道 RetSat 是多能干细胞中染色体凝聚的关键稳定剂。这突出了 RetSat 在早期胚胎发育中的重要作用，以及 RetSat 作为评估多能干细胞质量的有效生物标志物的潜在价值。
